II / CHROMATOGRAPHY / Paper Chromatography
analysis of sterol dehydration products from reRned
rapeseed oil (the composition of which is of
interest for detecting adulteration of other oils,
e.g. olive oil). Sample preparation consisted of
preparing a 1 : 5 dilution of the oil. The Rrst
LC column isolated the hydrocarbons (with the
column backSushed by a stronger eluent after each
analysis), while the second one separated the
products of interest into groups, such that the
closely related compounds could be separated by GC.
The fractions from LC-LC and the related gas
chromatograms are numbered.
Water Analysis
Brinkman, Vreuls, Noij and others have worked on
the enrichment of organic materials from water on
LC cartridges, followed by on-line liquid or thermal
desorption into a gas chromatograph. The aim is
a permanent, fully automated analysis of pesticides
and other critical contaminants in rivers or the supply
lines of water works. A standard procedure consists
in extraction of 1}10 mL of water on short polymer-
packed LC columns, which are then washed with
clean water and dried by a stream of nitrogen.
After desorption with ethyl acetate, the sample
is transferred through the on-column or loop-type
interface.
Conclusion
On-line LC-GC techniques are extremely powerful
with regard to selectivity, sensitivity (as a result of the
excellent clean-up) and efRciency (as most man-
ual sample preparation is integrated into the auto-
mated analysis). It seems, however, that currently
they are too demanding for widespread routine use.
Paper Chromatography
I. D. Wilson, AstraZeneca Pharmaceuticals, Mereside,
Alderley Park, Macclesfield, Cheshire, UK
Copyright ^ 2000 Academic Press
Introduction
The techniques of paper chromatography and paper
electrophoresis are sufRciently intertwined as to
be worth considering together, as indeed they often
were in books and reviews at the height of their
popularity.
397
See also: III/Crude Oil: Liquid Chromatography. Ter-
penoids: Liquid Chromatography. Essential Oils: Gas
Chromatography. Oils, Fats and Waxes: Supercritical
Fluid Chromatography. Petroleum Products: Liquid
Chromatography. Pesticides: Gas Chromatography.
Further Reading
Beens J and Tijssen R (1995) An on-line coupled HPLC-
HRGC system for the quantitative characterization of
oil fractions in the middle distillate range. Journal of
Microcolumn Separations 7: 345}354.
Grob K (1991) On-Line Coupled LC-GC. Heidelberg:
HuK thig.
Grob K (1994) On-line normal phase LC-GC. Methods for
routine applications. In: Riva del Garda Sandra P and
Devos G (eds). Proceedings of the 16th International
Symposium on Capillary Chromatography, pp. 1}9.
Heidelberg: HuK thig.
Grob K (1995) Development of the transfer techniques for
on-line HPLC-capillary GC (Review). Journal of
Chromatography 703: 265}276.
Grob K and Mariani C (1994) LC-GC methods for the
determination of adulterated edible oils and fats. In:
Tyman JHP and Gordon MH (eds), Development in the
Analysis of Lipids, p. 73. Cambridge: Royal Society of
Chemistry.
Kelly GW and Bartle KD (1994) The use of combined
LC-GC for the analysis of fuel products: a review. Jour-
nal of High Resolution Chromatography 17: 390}397.
Noij THM and van der Kooi MME (1995) Automated
analysis of polar pesticides in water by on-line SPE and
GC using the co-solvent effect. Journal of High
Resolution Chromatography 18: 535}539.
Vreuls JJ, de Jong GJ, Ghijsen RT and Brinkman UATh
(1994) LC coupled on-line with GC: state of the art.
Journal of the American Organization of Analytical
Chemist 77: 306}327.
Paper Chromatography
The origins of paper chromatography have been
traced back by some authorities as far as Pliny (23}79
AD), who described the use of papyrus impregnated
with an extract of gall nuts to detect ferrous sulfate.
Further examples of the use of paper chromatography
can be seen in the 19th-century work of the German
chemist Runge who described in his book Zur Far-
benchemie the use of this type of separation for the
investigation of inorganic mixtures. Subsequently
another book (Der Bildungstrieb der Stoffe) by
398 II / CHROMATOGRAPHY / Paper Chromatography
Runge appeared, containing examples of this work.
Further work in this area was undertaken by Schon-
bein and his student Goeppelshroeder, who investi-
gated the technique of Kapillaranalyse (capillary
analysis). However, these early studies seem to have
stimulated little real interest and, although there ap-
pear to have been some limited further studies in the
1930s and 1940s, it was not until the seminal work of
Consden, Gordon and Martin in 1944 on the analysis
of amino acids in protein hydrolysates, and sub-
sequent studies by Consden, Gorden, Martin and
Synge, that paper chromatography made a major
contribution to separations.
Paper chromatography is now obsolete, except per-
haps as an inexpensive technique for teaching
chromatography in schools and colleges. However
the introduction of paper chromatography may truly
be regarded as revolutionary, and was one of the
innovations in partition chromatography that led ul-
timately to the award of the Nobel prize to Martin
and Synge in 1952. One author stated, in a handbook
on the topic, that By this stroke of genius, they
changed the analysis of protein composition from a
lifetimes work to a 2}3-day simple technique that
could be carried out in any laboratory. So rapid was
the adoption of the technique that a book on the
subject published in 1954 contained nearly 4000 ref-
erences to its use. Quotations from textbooks of the
period contain statements such as Paper chromatog-
raphy is so widely used that it is impossible to make
more than a rough estimate of its application or it
can be stated that there is virtually no Reld of chem-
istry or biology in which paper chromatography has
not made a substantial contribution to the further-
ance of knowledge and understanding.
However, despite its huge impact at the time, paper
chromatography suffered from a range of prob-
lems that led to its rapid replacement by thin-
layer chromatography (TLC), to which it was inferior
in almost every respect. In particular, separations
on paper were often very slow (often up to 10 or
20 h), and spots tended to be much more diffuse
than, for example, separations on cellulose TLC
plates.
The Practice of Paper
Chromatography
Equipment
Probably the major advantage of paper chromatogra-
phy, and one that ensured its rapid adoption, is the
simplicity of the equipment required in order to per-
form it. Essentially this equipment is the same as that
now used for TLC and all that is required is a suitable
type of paper to act as the stationary phase, a means
of applying the sample, a developing tank and a sol-
vent system. A typical set-up for descending paper
chromatography is illustrated in Figure 1 showing, in
addition to the tank and solvent reservoir the anti-
siphon rod used to prevent excessive solvent Sow
from Sooding the paper. Tanks were normally oper-
ated with the atmosphere saturated with the vapours
of the solvent used for development in order to ensure
good and reproducible results.
Solvents
In general, the solvent systems used in paper
chromatography were based on mixtures of one or
more organic solvents with water. Acids (HCl, acetic,
etc.) or bases (aqueous ammonia) were added to
control the ionization of the analytes. Typical solvent
mixtures for amino acids, for example, might be
composed of butan-1-ol, acetic acid and water; butan
1-ol, pyridine and water or phenol and water. For
sugars, solvents based on ethyl acetate, pyridine and
water; ethyl acetate, propan-1-ol and water or ethyl
acetate, acetic acid and water were popular. For inor-
ganic ions, solvent systems such as acetone, concen-
trated HCl and water; pyridine and water or butan-1-
ol and HCl mixtures were suitable. In the case of
some of these solvents the mixtures suggested separ-
ated on standing into two phases. In such circum-
stances it was customary to separate the two phases
and use the aqueous layer to saturate the atmos-
phere in the developing tank and the organic layer
as the eluent for chromatography.
Papers
The media used for both paper chromatography and
paper electrophoresis were based on Rlter paper, with
Whatman no. 1 being perhaps the most widely used
and no. 3 also popular. However, paper manufac-
tured by other companies was also used and
Schleicher & Schull 2043b paper was popular accord-
ing to some sources. Paper suitable for use in
paper chromatography was also manufactured by
Munktell, Macherey Nagel, Eaton-Dikeman and
DArches. Not surprisingly, the availability of a range
of papers produced numerous studies comparing the
relative merits of the different products, eliciting
the somewhat jaundiced comment in one review of
the subject that These workers are not always in
agreement 2 and it is probable that the differ-
ences introduced by their individual experimental
methods are of a greater magnitude than the dif-
ferences between the various grades of paper. Some
papers were available in slow, standard and fast
grades with the speed of development controlled by
the coarseness of the cellulose Rbres and the packing
 II / CHROMATOGRAPHY / Paper Chromatography 399

Figure 1 A typical commercial system (Shandon Unikit) for descending chromatography. (A) Set-up for hanging the paper, which
hangs freely over the anti-siphon rod (1) and dips into the solvent reservoir where it is held in place with the anchor rod (2). (B) The
assembled reservoir and paper in place in the developing tank. At this point the solvent would be added and the tank closed with the lid.

density. In general, the standard papers gave the best
compromise between speed and resolution, with fast
papers more suitable for simple separations and the
slow papers used where the greatest resolution was
required. Whilst suitable for analytical work, these
papers were often replaced in preparative applica-
tions by more specialized materials such as Whatman
no. 3 MM and 31ET or Schleicher & Schull 2071. In
addition to pure cellulose, a variety of modiRed
papers were also produced, including ion exchange
materials, acetylated or benzoylated papers, silicone
oil-impregnated papers, as well as silica and alumina-
impregnated papers.
Modes of Paper Chromatography
In most of its practical aspects (e.g. sample applica-
tion, equipment such as developing tanks and visual-
ization procedures), paper chromatography some-
what resembles TLC. The most noticeable differ-
ence is that, as the paper is not rigid, it must either be
suspended from an appropriate support during devel-
opment or arranged in such a way as to be self-
supporting. Only the major types of paper
chromatography are described below, but it should
be noted that there were many minor variants of the
technique (e.g. centrifugal, continuous).
400 II / CHROMATOGRAPHY / Paper Chromatography
Figure 2 (A) One method of ascending chromatography involved the formation of a self-supporting cylinder, held together with
tongued clips. The cylinder was then placed in a tank containing the solvent for development (B).
Ascending Paper Chromatography.
In the ascending mode of development the paper is
suspended so that the lower edge is below the level of
the solvent, and the solvent moves up via capillary
action. An alternative to suspending the paper was to
form a self-supporting cylinder from the paper. These
arrangements are illustrated in Figure 2. As with
TLC, multiple development, with either the same or
different solvent, was used to improve resolu-
tion, although the time taken for each development
must have made this especially tedious to perform.
Descending Paper Chromatography
The descending method of chromatogram develop-
ment was that originally proposed by Martin and his
co-workers. In descending paper chromatography the
upper end of the paper is immersed in a solvent
contained in a suspended trough so that the Sow,
initiated as in the ascending mode by capillary action,
is sustained by gravity and will continue so long as
there is solvent to feed it. This had the useful conse-
quence that a sheet of any (practical) length could
be used. In addition, the solvent could be allowed to
run off the end of the paper, thus extending the
chromatographic run if needed to improve resolution,
or enabling compounds to be eluted from the paper
and collected for further experiments. The results
obtained for a particular sample/solvent system com-
bination run in either ascending or descending mode
were usually similar; however, the latter was gener-
ally faster.
Two-dimensional Separations on Paper
Where separations were not achieved in a single
development, it was often possible to achieve the
desired result using a second solvent system of
different composition and development in a sec-
ond dimension at 903 to the original direction of
chromatography. Two-dimensional paper chromato-
graphy was Rrst described by Consden, Gordon and
Martin for the separation of 20 amino acids, but was
subsequently widely employed. An additional possi-
bility was the use of paper chromatography in one
direction with paper electrophoresis (both high and
low voltage) in the second. Indeed, there are numer-
ous examples in the literature of either chromatogra-
phy followed by electrophoresis or electrophoresis
followed by chromatography. A typical example of
the type of result that could be obtained using two-
dimensional paper chromatography is shown in
Figure 3, whilst Figure 4 shows the combination of
electrophoresis followed by chromatography in the
second dimension for amino acids in fruit juice.
 II / CHROMATOGRAPHY / Paper Chromatography 401

Figure 3 A two-dimensional separation of a mixture of black and brown ink using butan-1-ol}ethanol}2 mol L\1 aqueous ammonia
(6 : 2 : 2) for the first development and butan-1-ol}acetic acid}water (6 : 1.5 : 2.5) for the second dimension, on Whatman no. 1 paper.
Key: 0, origin; 1, dark blue material remaining at or near the origin; 2, yellow pigment; 3, pink pigment; 4, diffuse brown pigment; 5, pink
pigment; 6, yellow pigment; 7, scarlet pigment; 8, pink pigment; 9 and 10, faint spots of orange and yellow pigments respectively.

Horizontal or Circular Paper Chromatography
Horizontal (or circular) paper chromatography was
performed in two ways. In the classical method,
a spot of the sample to be analysed was placed at the
centre of a circular Rlter paper. Then a short wick was
made by making parallel incisions c. 2 mm apart from
the edge of the Rlter paper to its centre. This wick was
then cut to an appropriate size and bent so that it
dipped into the solvent contained in a Petri dish. The
general arrangement is shown in Figure 5A. Sub-
Figure 4 A typical two-dimensional separation of amino acids
in orange juice effected by electrophoresis in pyridine}acetic acid
followed by chromatography with butan-1-ol}acetic acid}water for
the second. Detection with ninhydrin.
sequently, apparatus became available that elimi-
nated the need for cutting the paper to form a wick,
and one such is shown in Figure 5B.
Preparative Paper Chromatography
For a time preparative paper chromatography was an
important method for the isolation of substances,
leading to comments such as: These methods are so
well developed today that some laboratories prefer
them to the methods of column chromatography.
The simplest methods of preparative paper
chromatography were essentially scaled-up versions
of the analytical methodology using either several
sheets of paper or custom-made preparative card-
boards (e.g. Schleicher & Schull 2071). In addition,
techniques were developed such as the Chromatopile
(a number of discs of Rlter paper in a tightly com-
pressed stack to form a column, with development by
either ascending or descending chromatography), the
Chromatopack (strips or sheets of paper pressed to-
gether to form a block which was then developed as if
it were a single sheet) or rolls of Rlter paper wound
over a core of polyethylene and inserted into a poly-
ethylene column (sometimes these rolls were placed
in a pressurized jacket). Using such techniques the
preparation of milligram quantities of material was
readily achieved.
402 II / CHROMATOGRAPHY / Paper Chromatography
Figure 5 (A) Horizontal circular paper chromatography based
on the method devised by Rutter. The upper part of this diagram
shows 1, the paper; 2, the circle of sample applied to the paper
(this could also be in the form of individual spots of different
samples); and 3, the wick cut into the filter paper. The paper was
supported on a Petri dish (4) containing the solvent (5) into which
the wick was dipped in order to initiate development. Later the
methodology was adapted by the introduction of a special devel-
opment chamber which removed the need to cut a wick into the
paper. One such, based on the apparatus devised by Kawerau, is
shown in (B): 1, lid; 2, paper; 3, solvent capillary; 4, adjustable
collar; 5, solvent.
Applications of Paper
Chromatography
Given the importance of paper chromatography in
its heyday, a list of its applications covers all
types of analytes, including proteins, peptides,
amino acids, poly-, oligo-, di- and monosaccharides,
natural products, sterols, steroids, bile acids, pig-
ment, dyes and inorganic species. A typical applica-
tion to the separation of a series of inks is shown in
Figure 6.
Paper Electrophoresis
It is possible to trace the development of paper elec-
trophoresis back to the work of Konig, beginning in
1937 with a publication in Portugeuse. However, this
work attracted little interest at the time and it was the
Figure 6 Separation, by ascending paper chromatography, of
a series of ink samples (in duplicate). Key: RB, royal blue; Bk,
black; G, green; Br, brown; Sc, scarlet; O, origin; SF, solvent front.
Solvent system butan-1-ol}acetic acid}water (6 : 1.5 : 2.5) with
Whatman no. 1 paper.
later work of Wieland and Fischer on amino acids in
1948 and Durram on serum proteins (1949, 1950)
that attracted the attention of the scientiRc commun-
ity. Some measure of the importance of the technique
in its heyday may be gained from the observation in
a volume on paper chromatography and electrophor-
esis published in 1957 that more than 2000 papers
dealing with the subject of zone electrophoresis have
appeared. Over 90% of these have dealt with paper
electrophoresis. Faced with such apparent success, it
is possible to forgive the enthusiasm of the authors of
a subsequent manual, published in 1977, on the sub-
ject who felt able to say that: In fact, it can be truly
said that the history of paper electrophoresis still lies
before it. In fact, as with paper chromatography, this
type of electrophoresis is now considered by most
workers to be entirely obsolete.
The Practice of Paper Electrophoresis
Paper electrophoresis can be broadly divided into
three main techniques: low voltage, high voltage and
continuous. Of these, the low voltage (up to 1000 V)
technique was probably the most widely used.
Low Voltage Paper Electrophoresis
Strips of paper arranged either vertically or horizon-
tally and moistened with the buffer were used.
The application of the voltage (2}10 V cm\1) used to
perform separations generated some heat, but this
was generally carried away by evaporation when
the open strip method was used. In the open
strip technique the paper was suspended between the
 II / CHROMATOGRAPHY / Paper Chromatography
electrodes in the saturated gaseous phase of the devel-
oping chamber. This suspension was accomplished in
a wide variety of ways, with one review of the tech-
nique stating that: Paper has been arranged in this
chamber in almost every conceivable conRguration,
but it is usually either pulled horizontally taut or
allowed to hang free from a central support at the
apex. Both the horizontal and hanging strip methods
were reported to provide excellent resolution, but the
latter was claimed to give better reproducibility.
Other conRgurations included the semi-closed strip,
where the paper was supported on one side by
a cooled glass surface to enable temperature control,
and the closed-strip method where the paper was
either held between two glass plates or submerged in
a nonpolar immiscible liquid (e.g. heptane or carbon
tetrachloride). With the former system, evaporation
was not permitted and pressure could be applied
so as to control the amount of electrolyte taken up by
the paper. Using the nonpolar immiscible liquid
method, some heat was removed from the paper by
convection and conduction to a thermostatic bath.
A simple commercial low voltage paper electrophor-
esis apparatus, of the open strip type, is illustrated in
Figure 7.
As well as one-dimensional seperations, two-
dimensional paper electrophoresis was also performed
when needed to improve particular separations.
Figure 7 A simple commercial apparatus (Shandon Unikit) for paper electrophoresis. (A) The electrophoresis assembly showing the
application of the samples to the paper which is suspended in a V shape via a glass rod. (B) Once prepared, the assembly is placed in
the tank and the current switched on.
403
High Voltage Paper Electrophoresis
The name high voltage electrophoresis was used to
describe separations performed at voltages from 1 to
10 kV. The potential gradients used in high voltage
systems were generally in the region of 50}100 V cm\1
and, as a consequence, one of the main problems
encountered was heating. The apparatus used there-
fore required the presence of some form of heat ex-
changer to ensure that the heat was conducted away.
High voltage electrophoresis was considered to be
best used for low molecular weight substances with
many applications in amino acid analysis.
Continuous Electrophoresis
In continuous paper elecrophoresis the sample was
applied continuously to the paper (Figure 8), enabling
a considerable volume to be applied over time, allow-
ing preparative separations to be performed. The
layout of the paper in this type of separation is shown
in the diagram. Thus, the paper is suspended vertically
(often referred to as a curtain) so that the buffer
solution Sowed downwards (as in descending
chromatography). A Reld was then applied across the
direction of the Sow, causing the ionic substances to be
separated, as indicated in the Rgure. The individual
components of the mixture could be collected into
appropriate receptacles as they eluted from the paper.
404 II / CHROMATOGRAPHY / Paper Chromatography
Figure 8 The arrangement for continuous electrophoresis. (A)
Sample was applied continuously at this point and travelled down
the paper under the influence of the flow of buffer. Current was
applied at the point indicated by # and!, causing the compo-
nents (e.g. B) to separate. They could then be collected into
suitable receptacles as they eluted from the paper at C.
Applications of Paper Electrophoresis
As with paper chromatography, the applications of
paper electrophoresis were legion and included amino
acids, organic acids, natural products such as alkal-
oids, polysaccharides, nucleotides, proteins, peptides,
pigments and inorganic species. The scope of the
applications of paper electrophoresis is best appreci-
ated by reference to the texts indicated in the section
on Further Reading.
Detection and Quanti\cation of
Substances on Paper Chromatograms
or Electropherograms
As in TLC, following separation the papers are re-
moved from the developing chamber and dried. Col-
oured spots were visualized directly without difR-
culty, whilst those that Suoresce under UV irradiation
were also detected relatively easily. In the case of
colourless compounds, many of the visualization pro-
cedures, of varying degrees of speciRcity, currently
used for this purpose in TLC were also used for
detection after paper chromatographic separation,
using either spraying or dipping. Although considered
primitive by comparison with modern methods, sep-
arations on paper were also used for quantitative
assays in addition to qualitative work. As with other
planar methods, varying degrees of sophistication
were employed, from comparison of the size of spots
compared to a standard, cutting out the bands/spots
and eluting the analytes for subsequent spectroscopic
determination (i.e. UV, visible or Suorescence
measurements) all the way up to the use of den-
sitometry (with accuracies of $5%).
Conclusions
Paper chromatography and electrophoresis were once
techniques of considerable importance but this is no
longer the case. Whilst still useful as an aid to teach-
ing chromatography in schools and colleges, there are
virtually no situations where separations originally
developed for paper chromatographic methods can-
not now be performed faster and better by TLC. The
same comments apply to the relationship between
paper electrophoresis and the modern slab gel
technique.
Acknowledgement
Figures 1, 2, 4 and 7 are adapted from a Shandon
Southern Product Manual and are reproduced with
permission.
See also: I/Electrophoresis. II/Chromatography:
Thin-Layer (Planar): Densitometry and Image Analysis;
Historical Development; Spray Reagents.
Further Reading
Block, RJ, Durrum, EL and Zweig G. (1958) Paper
Chromatography and Paper Electrophoresis, 2nd edn.
New York: Academic Press.
Consden R, Gordon, AH and Martin AJP (1944) Qualitat-
ive analysis of proteins: a partition chromatographic
method using paper. Biochemistry Journal 38: 224.
Heftmann, E. (1961) Chromatography. New York: Rein-
hold.
Lederer E and Lederer M (1953). Chromatography.
Amsterdam: Elsevier.
Morris CJOR and Morris P (1964) Separation Methods in
Biochemistry. London: Pitman.
Pucar Z (1960) Kontinuierliche electrophorese und
zweidimensionale electrochromatographie. Journal of
Chromatography 4: 261.
Smith I and Feinberg JG (1977) A Manual for Paper and
Thin-layer Chromatography and Electrophoresis, 2nd
edn. Shandon Southern Products.
Stock R and Rice CBE (1967). Chromatographic Methods,
2nd edn. Gateshead on Tyne: Northumberland Press.